Sepsis remains a leading cause of death in critical care unit, and is also frequently associated with serious consequence such as multiple organ failure. Gram-negative bacterial endotoxin, also known as lipopolysaccharide (LPS), has been suggested to play a pivotal role in such septic complications (Houdijk et al, 1997). The acute phase plasma protein, LPS binding protein (LBP), binds circulating LPS to extract it from micelles, and transfer it to either soluble or membrane-bound CD14 receptor in monocytes and macrophages. The interaction of this complex with Toll-like receptors (TLRs) is thought to initiate intracellular-signaling reactions, via transcription factor NF-κB. Activation of protein kinases mediates the production of inflammatory cytokines, which contribute to septic shock. It has also been shown that in the absence of plasma LBP, the LPS is able to directly interact with CD14, yielding similar effects. Thus, treatment of endotoxaemia and sepsis would be greatly aided by blocking the activity of endotoxin and/or removing them from the body fluids of patients, as cationic peptides and analogues do (de Haas et al, 1998; Scott et al, 2000).
LPS from gram-negative bacteria induces the amoebocytes of limulus to aggregate and degranulate. This response underlies the important defense mechanism of limulus against invasion of gram-negative bacteria (Ding et al, 1995). As a molecular biosensor, Factor C can be autocatalytically activated by femtograms of LPS to trigger the coagulation cascade (Ho, 1983), suggesting that it contains high affinity LPS-binding domains. Recently, two regions of Factor C that exhibit exceptionally high LPS binding affinity were defined as the Sushi1 and Sushi3 domains (Tan et al, 2000a). Two 34-mer chemically synthesized peptides, S1 and S3, spanning the 171-204 and 268-301 amino acid residues of Factor C (GenBank Accession No. S77063), are derived from Sushi1 and Sushi3 domains, respectively. (The S3 peptide consisting of residues 268-301 of Factor C is shown in SEQ ID NO:1). Both peptides inhibit LPS-induced limulus amoebocyte lysate (LAL) reaction and LPS-induced hTNF-α secretion (Tan et al, 2000b). See also U.S. Pat. No. 6,719,973, the entire content of which is herein incorporated by reference. The application value of these two peptides would be boosted if they could be obtained by cost effective and large-scale methods such as recombinant expression in prokaryotic systems. However, expression of smaller peptides tends to encounter technical difficulties (Le et al, 1991; Latham, 1999).
Trace levels of endotoxin or lipopolysaccharides (LPS) cause pathophysiological reactions such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock and death. Intensive research is being carried out to develop more sensitive techniques that are able to remove minute levels of endotoxin from pharmaceutical fluids to meet higher standards of safety (Petsch 2000). Adsorption methods have proven to be the most effective (Minobe 1982) in removing endotoxins from solutions and many methods have been developed for different target solutions with varying efficiencies. However, most of these methods are not efficient over a wide range of pH and ionic strength (Petsch 2000). In addition, there is always a compromise between protein recovery and LPS removal, such that the clearance factor is often disappointing when the LPS feed concentration is low.